Title of Invention

A PROCESS FOR LOW MOLECULAR WEIGHT CHITOSAN

Abstract

A process for preparation of low molecular weight chitosan, which comprises a. dissolving chitosan of higher molecular weight in dilute aqueous acidic medium by stirring, b. characterized in that adding an aqueous persulphate solution of concentration in the range 0.5 to 2 mM with continued stirring for a period of 1 to 5 hrs at a temperature in the range of 40 to 80°C, c. sedimenting the reaction mixture by its cold storage at a temperature in the range of 0-10°C for a period of 12 to 14 hrs, d. separating the sediment by known method , e. precipitating of the filtrate with alcohol to get low molecular weight chitosan as precipitate.

Full Text

The present invention relates to a process for preparation of low molecular weight chitosan. The invention, in particular describes the preparation of low molecular weight chitosan and chitooligomers, which are of high value addition for use in medicine, agriculture, food and non-food industries as well as in other allied applications. Chitin, a ß 1,4-linked polymer of N-acetyl-D-glucosamine, is the second most abundant natural biopolymer derived from exoskeletons of crustaceans and also from cell walls of fungi and insects. Chitosan, a copolymer of ß 1,4-2-acetamido-2-deoxy-D-glucose (-20%) and ß 1,4-2-amido-2-deoxy-D-glucose (80%) units, is a product derived from de-N-acetylation of chitin in the presence of hot alkali. Chitosan, is infact a collective name representing a mixture of de-N-acetylated chitins, deacetylated to different degrees. In India, chitosan availability will be more than 300 metric tonnes annually. Both chitin/chitosan and their modified derivatives find extensive applications in medicine, agriculture, food and non-food industries as well. Reference may be made to G.S.Braek, T. Anthonsen and P.A.Sandford (Eds.), Chitin and chitosan, Elsevier Applied Science, NY, 1989, for an extensive coverage of chemistry, biochemistry and functional aspects of chitin/ chitosan. All these are the results of their biological activity, biocompatibility and biodegradability in combination with their low toxicity. However, one major constraint in their efficient usage is the high molecular weight of these polymers, which limits their solubility in aqueous medium. High viscosity, even at low concentrations of chitosan is another limiting factor in its use for specific applications. The functional properties of chitosan and its oligomers are mainly dependent upon their molecular weight and the consequent ease of solubility in aqueous media. Nevertheless, preference is given for using low molecular weight chitosans, having high solubility in aqueous acidic or neutral medium, for some specific physiological applications such as antibacterial activity, hypocholesterolemic activity, antitumor activity, antihypertensive activity and immunopotentiating activity in medicine and agriculture. Increasing attention has recently been given to converting chitin and chitosan to chitooligosaccharides, which are shown to exhibit a variety of useful functional and biopharmacological attributes. Low molecular weight chitosan and chitooligomers can be prepared by acidic or enzymic degradation of polymeric chitosan molecule. The enzymic process is generally preferable over that of acid degradation, because the course of hydrolysis and product distribution can be controlled precisely. Reference may be made to J.A.Rupley, Biochim. Biophys. Acta, 83 (1964) 245-255, wherein the hydrolysis of chitin/chitosan in strong hydrochloric acid was studied over a range of acid cocentration, temperature and duration. The drawback of the process is that large quantities of monomeric glucosamine appeared in the early hydrolysates and the concentrations of dimer, trimer and higher oligosaccharides were never obtained in comparable concentrations. Further, the inherent difficulties of removing the acid by alkali neutralization followed by desalting by chromatographic means make the whole process expensive and very laborious, and therefore not viable for commercial exploitation. These drawbacks have been partially overcome by using enzymatic method, which also minimizes alterations in the chemical nature of reaction products. Reference may be made to K.Sakai, T.Uchiyama, Y.Matahira and F.Nanjo, J. Ferment. Bioeng., 72 (1991) 168-199; M.Charpentier and P.Percheron, Int.J.Biochem., 15 (1983) 289-292 for a few enzymic procedures for the preparation of low molecular weight chitosans and water soluble chitooligosaccharides. Chitinases and chitosanases, which are widely distributed in fungi, bacteria and plants, are the specific enzymes used for this purpose, although reference may be made to M.Yalpani and D.Pantaleone, Carbohydr. Res., 256 (1994) 159-173, who have used several of the commercial enzymes such as lipases , proteases and glycanases for non-specific hydrolysis of chitin, chitosan and their derivatives. The drawback of using such enzymically hydrolysed chitosan preparations for biomedical and food purposes is the undesirable level of chitosan pyrogenicity caused by the presence of about 0.1% (w/w) of the proteins of the enzyme complex. This has been partially overcome by enzyme immobilization on a suitable matrix. Reference may be made to A.V.IIyina, V.E.Tikhonov, A.I.AIbulov and V.P.Varlamov, Process Biochem., 35 (2000) 563-568, for a chitinolytic enzyme complex produced by Streptomyces kurssanovii immobilized on macroporous crosslinked chitosan, for the preparation of acid-free water soluble chitosan with a low molecular weight. In this reference a two step hydrolysis was used, the first hydrolysis at pH 4.6 giving rise to a 22- 24 kDa chitosan of low solubility in water, whereas the second hydrolysis at pH 6.2 gave a low molecular weight (2-9 kDa) chitosan, which was easily water soluble. The main drawbacks of this enzymic procedure are, 1. The method involves hydrolysis by an enzymic complex in batch reactors, which may not be cost effective, 2. The method includes enzyme immobilization step, adding to additional cost and time involved in its preparation as well as time required for desorption of the hydrolysed products, and 3. the method necessitates too many steps and subsequent follow up, which make the whole process, laborious, time consuming and not cost effective. Reference may be made to Japanese patent No. JP 03277277, entitled Novel chitosanase, bacterium for producing the same and preparation of low molecular weight chitosan, wherein low molecular weight chitosan useful as a coagulant or solubility accelerating agent and not containing an excessive amount of salt is prepared by treating chitosan with a specific bacterial chitosanase and subsequently separating the derived chitosan oligosaccharide. The main drawback of this method is utilization of expensive chitosanase enzyme, which is not available in bulk for commercial exploitation. Reference may be made to Japanese patent No. JP 09031104, entitled Production of low molecular weight chitosan and chitooligosaccharide, which are produced by hydrolysis with strong acid. The drawbacks of this method are the use of strong mineral acid which results in random hydrolysis of chitosan resulting in lower yields of products, and use of H2O2 for decolorization, Ca(OH)2 for neutralization of acid and alcoholic extraction of the byproducts. Reference may be made to Japanese patent No. JP 02011601, entitled Production of low molecular weight chitosan, wherein the molecular weight reduction of high molecular weight chitosan is effected by reacting with H2O2 under an acidic condition. The drawback of this method is the use of acid and H2O2, which may lead to random cleavage owing to non-specific oxidation especially at the amino group and as a result unidentifiable oxidized products may contaminate the low molecular weight chitosan making it unsafe for use in food, feed and medicine. Reference may be made to an American patent No. US 05705634, entitled High yield preparation of dimeric and decameric chitin oligomers, wherein the title compounds are produced by fragmentation and partial acid hydrolysis. Although claim is made for high yields of higher chitooligomers, the drawback is the use of acid for hydrolysis and its subsequent neutralization, and purification of the products. All these add to more number of steps involved for production, and as a whole the method may not be economical. Reference may be made to Japanese patent No. JP 05320204, entitled Production of N-acetylchitooligosaccharide, wherein chitosan is decomposed with chitosanase derived from Bacillus No.7-M followed by enzyme inactivation and product purification by gel filtration, electrodialysis and lyophilization. The drawback of this method is the use of expensive enzyme and laborious steps involved in product purification. Reference may be made to Japanese patent No. JP 09031105, entitled Production of low molecular weight chitosan and chitooligosaccharide, for producing the title comopounds by irradiating a solution of chitosan with ultrasonic waves. The drawback of this method is low recovery of the products and use of costly ultrasonic generator for effecting polymer degradation. The main object of the present invention is to provide a process for low molecular weight chitosan, which obviates the drawbacks discussed above. Another object of this invention is to prepare a range of low molecular weight and modified chitosans for a variety of end use applications in various fields, including food, agriculture and medicine. Yet another object of the present invention is to recover chitooligomers having high potential biomedical uses. The novelty in the present invention is that the degradation of chitosan molecule is induced by free radical initiation at the amino group of the glucosamine residues followed by subsequent glycosidic cleavage giving rise to low molecular weight chitosan. The probable reaction mechanism involved the following steps. (Figure Removed) Accordingly the present invention provides a process for preparation of low molecular weight chitosan, which comprises a. dissolving chitosan of higher molecular weight in dilute aqueous acidic medium by stirring, b. characterized in that adding an aqueous persulphate solution of concentration in the range 0.5 to 2 mM with continued stirring for a period of 1 to 5 hrs at a temperature in the range of 40 to 80°C, c. sedimenting the reaction mixture by its cold storage at a temperature in the range of 0-10°C for a period of 12 to 14 hrs, d. separating the sediment by known method , e. precipitating of the filtrate with alcohol to get low molecular weight chitosan as precipitate. In an embodiment of the invention the aqueous acidic medium used is selected from dilute acetic acid, dilute lactic acid and a combination thereof. In an embodiment of the invention the concentration of the aqueous acidic medium used is 0.5 to 2%. In an embodiment of the invention the aqueous persulphate solution used is selected from potassium persulphate and ammonium persulphate. In an embodiment of the invention the reaction is performed by continuous stirring under inert conditions such as nitrogen atmosphere. In an embodiment of the invention the precipitation is effected by using alcohol selected from the group consisting of ethanol, methanol and isopropanol. In an embodiment of the invention the precipitate obtained is low molecular weight chitosan with molecular weight in the range of 37,000 to 40,000 Da. The preparation of low molecular weight chitosan and chitooligomers is accomplished as described in Scheme 1 (Scheme Removed) The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention Example 1. Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with continued stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 2. Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (0.37 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction "mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 3 Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (0.74 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 4. Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (1.85 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to molecular weight of about 40,000 Da and this was designated as low molecular weight chitosan. The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 5. Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 45°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 6. Chitosan (1 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 80°C. The reaction mixture was then cooled to supernatant was added isopropanol (2 volumes ) and the precipitate was separated by centrifugation. The precipitated material (yield, 41%), dissolved in water, was subjected to high pressure size exclusion chromatography which indicated a molecular weight of about 40,000 Da and this was designated as low molecular weight chitosan. The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 7. Chitosan (2 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring . An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 8. Chitosan (3 g) of molecular weight 96,000 Da was dissolved in 100 ml of 1% aqueous acetic acid by stirring. An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about Example 9. Chitosan (1 g) of molecular weight 1,50,000 Da was dissolved in 100 ml of 1% acetic acid by stirring. An aqueous solution of potassium persulphate (1.45 mM) was added to chitosan solution with stirring under N2 atmosphere for 2 hrs at 65°C. The reaction mixture was then cooled to The alcoholic supernatant was concentrated to 5 ml by rotary flash evaporation under reduced pressure at a bath temperature of about as chitooligomers as it contained a mixture of glucosamine and chitooligosaccharides with molecular weight in the range of 180 to 2,000 Da, in various proportions. Characterization of the products The LMWC was characterized by HP-SEC which gave a single peak indicating its homogeneity and having a MW in the range 37-40,000 Da (Fig. 1). CP-MAS 13C-NMR and IR data showed no spectral modifications, except a change in the polymorph (Figs. 2 and 3). HPLC of the chitooligomeric fraction showed (GlcNH2)5 and (GlcNH2)6 species in high abundance (Fig. 4). The main advantages of the present invention are: 1. The low molecular weight chitosan as well as the chitooligomers are easy to prepare. Their easy water solubility offers great advantages for different end use applications. 2. Neither costly reagents such as specific enzymes, buffer salts, etc. nor sophisticated equipments such as membrane bio-reactors and other gadgets are used in this process invention, which indirectly adds to cost effectiveness of the products obtained. 3. The percent recovery of low molecular weight chitosan is high, it is pure (>95%) and it is readily soluble in water without imparting any abnormal viscosity gain. 4. Both low molecular weight chitosan as well as chitooligomers have innumerable functional and biomedical attributes of considerable utility and the present process invention offers an easy means of producing them in bulk. 5. The products (yield: LMWC around 45% and chitooligomers around 5%) obtained are stable. They do not lose their functional and other attributes even after storage at ambient or refrigerated temperatures. We Claim: 1. A process for preparation of low molecular weight chitosan, which comprises a. dissolving chitosan of higher molecular weight in dilute aqueous acidic medium by stirring, b. characterized in that adding an aqueous persulphate solution of concentration in the range 0.5 to 2 mM with continued stirring for a period of 1 to 5 hrs at a temperature in the range of 40 to 80°C, c. sedimenting the reaction mixture by its cold storage at a temperature in the range of 0-10°C for a period of 12 to 14 hrs, d. separating the sediment by known method , e. precipitating of the filtrate with alcohol to get low molecular weight chitosan as precipitate. 2. A process as claimed in 1 wherein the aqueous acidic medium used is selected from dilute acetic acid, dilute lactic acid and a combination thereof. 3. A process as claimed in 1 and 2 wherein the concentration of the aqueous acidic medium used is 0.5 to 2%. 4. A process as claimed in 1 to 3 wherein aqueous persulphate solution used is selected from potassium persulphate and ammonium persulphate. 5. A process as claimed in 1 to 4 wherein the reaction is performed by continuous stirring under inert conditions such as nitrogen atmosphere. 6. A process as claimed in 1 to 6 wherein precipitation of step (e) is effected by known methods and by using alcohol selected from the group consisting of ethanol, methanol and isopropanol. 7. A process for the preparation of low molecular weight chitosan substantially as herein described with reference to the examples, and drawings accompanying this specification.

Documents:

433-del-2001-abstract.pdf

433-del-2001-claims.pdf

433-del-2001-correspondence-others.pdf

433-del-2001-correspondence-po.pdf

433-del-2001-description (complete).pdf

433-del-2001-drawings.pdf

433-del-2001-form-1.pdf

433-del-2001-form-18.pdf

433-del-2001-form-2.pdf

433-DEL-2001-Form-3.pdf